Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Apr;182(8):2262-8.
doi: 10.1128/JB.182.8.2262-2268.2000.

Complex function for SicA, a Salmonella enterica serovar typhimurium type III secretion-associated chaperone

S C Tucker  1 , J E Galán
Affiliations

Complex function for SicA, a Salmonella enterica serovar typhimurium type III secretion-associated chaperone

S C Tucker et al. J Bacteriol. 2000 Apr.

Abstract

Salmonella enterica encodes a type III secretion system within a pathogenicity island located at centisome 63 that is essential for virulence. All type III secretion systems require the function of a family of low-molecular-weight proteins that aid the secretion process by acting as partitioning factors and/or secretion pilots. One such protein is SicA, which is encoded immediately upstream of the type III secreted proteins SipB and SipC. We found that the absence of SicA results in the degradation of both SipB and SipC. Interestingly, in the absence of SipC, SipB was not only stable but also secreted at wild-type levels in a sicA mutant background, indicating that SicA is not required for SipB secretion. We also found that SicA is capable of binding both SipB and SipC. These results are consistent with a SicA role as a partitioning factor for SipB and SipC, thereby preventing their premature association and degradation. We also found that introduction of a sicA null mutation results in the lack of expression of SopE, another type III-secreted protein. Such an effect was shown to be transcriptional. Introduction of a loss-of-function sipC mutation into the sicA mutant background rescued sopE expression. These results indicate that the effect of sicA on sopE expression is indirect and most likely exerted through a regulatory factor(s) partitioned by SicA from SipC. These studies therefore describe a surprisingly complex function for the Salmonella enterica type III secretion-associated chaperone SicA.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Effect of a loss-of-function mutation in sicA on the intracellular and extracellular levels of SipB and SipC. Whole-cell lysate and culture supernatant proteins from wild-type S. enterica serovar Typhimurium, its isogenic sicA mutant strain SB221, or the same mutant carrying a sicA-complementing plasmid pSB814 (psicA) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a mixture of SipB and SipC monoclonal antibodies.
FIG. 2
FIG. 2
Absence of SicA results in decreased SipB and SipC stability. Wild-type S. enterica serovar Typhimurium and the isogenic sicA mutant strain SB221 were pulse-labeled with [35S]methionine for 2 min and chased with cold methionine for 60 min. At the indicated time points, samples were removed, immunoprecipitated with anti-SipB and anti-SipC polyclonal antibodies, and run on an SDS-PAGE gel. Labeled proteins were visualized by fluorography.
FIG. 3
FIG. 3
Effect of a loss-of-function mutation in sicA on the expression of sipC. The levels of C2,3D in the S. enterica serovar Typhimurium strain SB227, which carries an sipC::xylE gene fusion, and in its derivative strain SB266, which carries a loss-of-function mutation in sicA, were measured as described in Materials and Methods. Activity is expressed as percentage of wild-type activity, which was considered 100.
FIG. 4
FIG. 4
Effect of loss-of-function mutation in sicA on levels of type III secreted proteins InvJ, SipA, and SipD. Whole-cell lysate and culture supernatant proteins from wild-type S. enterica serovar Typhimurium and isogenic sicA mutant derivatives were probed for the presence of the different proteins, as indicated by Western immunoblotting with antibodies directed to the proteins or their epitope tags as appropriate (see Materials and Methods). Lane control, sample from an otherwise identical S. enterica serovar Typhimurium strain that does not express the M45-SipD epitope-tagged protein.
FIG. 5
FIG. 5
Interaction of SicA with SipB and SipC. Whole-cell lysates of the S. enterica serovar Typhimurium strain SB319 which expresses a functional, chromosomally encoded, M45-epitope-tagged form of SicA were immunoprecipitated with antibodies directed to SipB, SipC, or a preimmune serum (Pre), as described in Materials and Methods. Proteins that bound to the beads (P) or that remained in the supernatant (S) were probed by Western immunoblotting for the presence of M45-SicA with a monoclonal antibody directed to the M45 epitope.
FIG. 6
FIG. 6
SipB is stable and secreted in a sicA sipC double-mutant strain. Culture supernatants and whole-cell lysates of wild-type S. enterica serovar Typhimurium and several isogenic mutant strains were probed for the presence of SipB and SipC by Western immunoblotting with monoclonal antibodies directed to these proteins.
FIG. 7
FIG. 7
Culture supernatant protein profiles of wild-type and isogenic mutant strains of S. enterica serovar Typhimurium. Culture supernatants proteins from the different strains of S. enterica serovar Typhimurium were separated by SDS-PAGE and visualized by Coomassie blue staining, as described in Materials and Methods.
FIG. 8
FIG. 8
Effect of a loss-of-function mutation in sicA on the expression of sopE. The levels of β-galactosidase in the S. enterica serovar Typhimurium strain SB876, which carries a sopE::lacZ gene fusion, and its isogenic derivative strains SB879 and SB1235, which carry loss-of-function mutations in sicA or in sicA and sipC, respectively, were measured as described in Materials and Methods. Activity is expressed as percentage of wild-type activity, which was considered 100. As negative controls, levels of β-galactosidase were measured in the S. enterica serovar Typhimurium strain SB804, which carries a sitB::lacZ gene fusion (wild type), and its isogenic derivative strains of SB1234, which carry loss-of-function mutations in sicA.
See this image and copyright information in PMC

References

    1. Baudry B, Kaczorek M, Sansonetti P J. Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri. Microb Pathog. 1988;4:345–357. - PubMed
    1. Bergman T, Hakansson S, Forsberg A, Norlander L, Macellaro A, Baeckman A, Boelin I, Wolf-Watz H. Analysis of the V antigen lcrGVH-yopBD Operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV. J Bacteriol. 1991;173:1607–1616. - PMC - PubMed
    1. Cheng L W, Anderson D M, Schneewind O. Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica. Mol Microbiol. 1997;24:757–765. - PubMed
    1. Collazo C, Galán J E. The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol Microbiol. 1997;24:747–756. - PubMed
    1. Collazo C, Galán J E. Requirement of exported proteins for secretion through the invasion-associated Type III system in Salmonella typhimurium. Infect Immun. 1996;64:3524–3531. - PMC - PubMed

Publication types

Substances

LinkOut - more resources